The integrity and proper function of all epithelial tissues critically depends on E-cadherin- mediated cell-cell adhesion. Lack of E-cadherin causes severe developmental defects and is linked to tumor development. The recent discovery of E-cadherin as a mechanosensor raises the question of how mechanical forces regulate cell-cell adhesions in a physiological context. The long term goal of the project is to uncover the mechanisms by which mechanical forces regulate multi-cellular organization. Here, we propose to determine how cell-generated forces and local stiffness coordinately regulate E-cadherin adhesions at a sub-cellular level and lead to specific downstream mechanotransduction events. Firstly, a mixture of nascent and mature E-cadherin adhesions are essential to maintain dynamic cell-cell contacts, but the micro-scale organization of E-cadherin adhesions and the forces transmitted through them are difficult to discern within the complex surface that comprises native cell-cell contacts. In Specific Aim 1, we propose to demonstrate a biomimetic E-cadherin specific adhesion interface between an MDCK epithelial cell and an oriented layer of E-cadherin bound to a flexible polyacrylamide gel that enables (i) the spatial segregation of nascent and mature adhesions, (ii) measurement of forces transmitted through these adhesions via high-resolution traction force microscopy and (iii) switching of the interface from having predominantly mature to predominantly nascent E-cadherin adhesions by inhibition of formin-mediated F-actin nucleation. Secondly, it is unclear as to how cell-cell adhesion is impaired in some types of cancers even though E-cadherin expression stays largely undiminished. In light of that fact that epithelial cancer cells are significantly softer than normal epithelial cells, in Specific Aim 2, we will test the hypothesis that E-cadherin adhesion is coordinately regulated by the stiffness of the microenvironment and the cell's internal contractility. We will use polyacrylamide gels of cell-like stiffness and partial myosin inhibition to test this hypothesis. Finally, it has been proposed that enhanced force transmission via E-cadherin leads to enhanced vinculin recruitment via its binding to stretched a-catenin. In Specific Aim 3, we will quantitatively test this hypothesis in cells by quantifying the localization of GFP-tagged vinculin as well as the forces transmitted through nascent and mature E-cadherin adhesions in normal MDCK cells as well as MDCK cells in which a-catenin has been stably knocked down. Results of the proposed project will be crucial in systematically assessing the role of mechanical forces in E-cadherin adhesion and mechanotransduction. Knowledge gained will be essential to understand the functional basis of the role of E-cadherin in mediating epithelial tissue integrity, mechanical coherence and its dysregulation in disease states like cancer.